*2.3. Enzymatic Digestion*

In TESE, the goal of mechanically shredding tissues is to free the germ cells that are trapped in seminiferous tubules or adhered to the tissue. However, digestion of tissues using enzymes can be expected to facilitate the release of gametes by loosening the cellular contacts in the tubular wall. In fact, collagenase has been shown to provide sufficient dissolution in the tissue without decreasing cell viability [25] (Table 1). This was also confirmed by Salzbrunn et al. for cryopreserved testicular tissue, and enzymatic preparation of tissues using collagenase type AI provided high yields of vital spermatids and spermatozoa [26]. Subsequently, Fischer et al. reported the first pregnancy using spermatozoa extracted using the same method in ICSI [27]. However, when compared to type AI, collagenase type IV has been shown to be more effective in isolation and recovery of viable spermatozoa and round spermatids from testicular samples [28]. Type IV collagenase is preferred for the processing of testicular tissue, since type IV collagen is specifically localized in the basement membrane of the seminiferous tubules and within the extracellular matrix (ECM) layers [29]. On the other hand, collagenase type IV is produced by Sertoli cells (SCs), and its target (collagen IV) has been shown to affect the dynamics of SC-tight junctions, which mediates translocation of preleptotene and leptotene spermatocytes residing at the basal compartment of the seminiferous epithelium into the adluminal compartment for further development [30]. However, the exact mechanisms regulating the events of spermiation and sperm release have not ye<sup>t</sup> been clearly elucidated. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), which belong to the M12 metallopeptidase family, are responsible for the migration of differentiated germ cells through the seminiferous tubule wall by organizing the degradation and reformation of the ECM [31]. In fact, we recently demonstrated that ADAMTS1 and ADAMTS5 protein levels expressed in Sertoli cells were decreased approximately 2-fold in cases of NOA [32]. The effect of enzymatic digestion on the success of sperm retrieval in cases where these proteases are defective is a subject of further research. For these reasons, the use of type IV collagen has been widely preferred to increase sperm recovery rates, because of both its disrupting effect on the integrity of the tubule basement membrane and its ability to break the connections of germ cells with Sertoli cells [33]. During the process, clotting caused by free DNA released from dead cells can be prevented by the addition of DNAase enzyme [28].

Since enzymatic digestion of testicular tissue allows obtainment of an isolated germ cell suspension free of tissue artifacts, its efficiency is higher compared to rough mechanical separation, which causes contamination with large amounts of damaged cells, free nuclei, and residual tissue fragments in the cell suspension. Indeed, spermatozoa retrieval rates (SRRs) between 7% and 33% were reported following enzymatic processing of tissue suspension in TESE cases in which no spermatozoa could be obtained by mechanical mincing [33–36]. The differences between the SRRs reported in the studies may be due to the experience of the embryologists, the histopathological status of the testicular tissues, and variations in the number of cases. In particular, the time spent and effort expended during the mechanical shredding of seminiferous tubules have significant effects on the chance of sperm detection in the laboratory. The crucial role of laboratory handling, especially in the managemen<sup>t</sup> of compromised testicular specimens, has been discussed previously [37]. Similarly, Modarresi et al. reported serum follicle-stimulating hormone (FSH) and luteinizing hormone levels as factors that may affect SRRs after enzymatic digestion treatment [33]. On the other hand, collagenase treatment has been shown to increase the cytokine population in the testis by stimulating an immune response, but its effects on spermatozoa remain unclear [38]. In general, processing with enzymatic digestion from testes in which very few spermatozoa are predicted to be present should be considered an effective practice.


**Table 1.** Clinical results of enzymatically tissue processing methods for sperm recovery by TESE.
